Research: In recent years, intensive effort has focused on the investigation of biological approaches for the treatment of cancer, including immunotherapy utilizing cytokines, antibodies and/or vaccines, gene therapy as well as inhibition of tumor angiogenesis among others. Although immunotherapeutic approaches such as systemic administration of interleukin-2 (IL-2) have provided meaningful benefit to some patients with metastatic renal cell carcinoma or melanoma, many questions remain regarding the best approach to maximize the potential efficacy of biological therapy. Given the complexity of signals engaged during the host antitumor immune response, and the intricate network of interactions within the tumor microenvironment, it appears that much as occurred with the clinical evolution of combination chemotherapy, the full potential of biological therapies for cancer will most likely be realized using rationally-designed combinations of agents with complementary mechanisms of action. Laboratory studies: We investigate molecular mechanisms by which the host immune response may be engaged to induce alterations in the tumor microenvironment to effect disease regression (i.e. modulation of tumor neovascularization, induction of tumor and/or endothelial apoptosis, etc.), and also use these observations to facilitate the design of novel biologically-targeted treatment strategies for neuroblastoma and/or renal cell carcinoma. Much of our recent effort has focused on investigation of the antitumor activity of two cytokine-based combinations, IL-12/pulse IL-2 and IL-18/IL-2, and delineation of the respective mechanisms which mediate their therapeutic efficacy. In mice bearing well-established primary and/or metastatic neuroblastoma or renal cell carcinoma tumors, systemic administration of IL-12/pulse IL-2 can induce complete durable tumor regression in 80% or more of treated mice. Comparable responses are achieved after treatment with IL-18+/- IL-2. These studies have now defined several of the critical mechanisms by which these therapies can modulate the local tumor microenvironment to induce disease regression. Notably, IL-12/pulse IL-2 induces rapid vascular endothelial injury with tumor and/or endothelial apoptosis, inhibits tumor neovascularization and mediates CD8+ T cell-dependent tumor regression via mechanisms which share a common dependency on IFN-gamma and the Fas/Fas-L apoptosis pathway. Collectively, these observations suggest a mechanism whereby CD8+ FAS-L+ T cells may infiltrate the local tumor site and interact with Fas+ vascular endothelial and/or tumor cell populations to induce apoptosis, inhibition of angiogenesis, and ultimately, overall tumor regression. In mice bearing neuroblastoma tumors, IL-12 not only upregulates proapoptotic pathways within the tumor cell compartment, but also downregulates prosurvival factors that may otherwise confer an intrinsic resistance to apoptosis. Similar but distinct mechanisms may be engaged by combined administration of IL-18/IL-2. More recent studies from our laboratory have now investigated the antitumor activity and mechanisms of action by IL-12 related cytokines including IL-23 and IL-27, and have demonstrated that these cytokines mediate potent antitumor effects in preclinical models of murine neuroblastoma and renal cell carcinoma. Our studies suggest that these cytokines may engage novel antitumor mechanisms and be rationally combined with existing antitumor cytokines such as IL-2 and/or IL-12 as well to achieve an improved spectrum of therapeutic efficacy. Rigorous preclinical investigation of the therapeutic efficacy and mechanisms of action by new therapeutic approaches necessitates the use of clinically relevant, therapeutically-challenging model systems, and the ability to monitor and/or quantitate various features of tumor biology in vivo. Towards this end, we have established unique preclinical models of transplantable orthotopic intraadrenal murine neuroblastoma using existing cell lines, and have developed and characterized a panel of novel transplantable neuroblastoma cell lines derived from spontaneous tumors arising in our colony of N-myc transgenic mice (obtained via a collaboration with Dr. William Weiss, UCSF). Further, to address the role of tumor versus host cell (i.e. endothelial) IFN-gamma responsiveness to biological therapies such as IL-12/IL-2, these N-myc transgenic mice also have been bred with relevant knockout strains to establish N-myc transgenic-knockout murine hosts and/or novel derivative neuroblastoma cell lines with targeted disruption of the gene encoding the IFN-gamma receptor. To complement these models, we have now established unique fluorescence-based approaches that allow for monitoring and/or quantitating the growth, neovascularization, metastasis and apoptosis of neuroblastoma tumors in vivo. In other studies, we are investigating the angiogenic phenotype and basic mechanisms governing the neovascularization of neuroblastoma tumors. We have demonstrated marked constitutive expression of vascular endothelial growth factor (VEGF) and FLT-1/FLK-1, angiopoietin-1 and TIE-2, as well as the matrix metalloproteinases, MMP-2 and MMP-9 by both murine and human neuroblastoma tumors and cell lines. Further, although treatment with immune-based therapies such as IL-12/pulse IL-2 inhibits the neovascularization of neuroblastoma tumors, there is no apparent modulation of the expression of various proangiogenic mediators and/or their receptors within these tumors. These observations suggest that targeted antagonists of proangiogenic mediators such as VEGF could play a role in the treatment of neuroblastoma , and that the efficacy of antiangiogenic immunoregulatory cytokines such as IL-12 might be expanded by combined administration with targeted antagonists of proangiogenic mediators such as VEGF-a proposed immunoangiostatic approach. Preclinical studies are now in progress to investigate this strategy. Clinical Investigation: Based on the potent efficacy of IL-12/pulse IL-2 in preclinical tumor models, we subsequently designed and executed a primate toxicology evaluation of the safety of this combination in cynomolgus macaques, and are performing a phase I investigation of IL-12/pulse IL-2 in adults with advanced solid tumors. Guided by our preclinical observations, hypothesis-driven translational studies have been incorporated into this clinical trial to investigate antitumor mechanisms engaged by the administration of IL-12/pulse IL-2 in humans. These include characterization of the functional immunoregulatory effects of IL-12/pulse IL-2 in vivo, prospective quantitative assessment of the antivascular activity of IL-12/pulse IL-2 utilizing dynamic-enhanced MRI (DEMRI) scans, as well as molecular and histopathologic investigation of antitumor mechanisms induced by IL-12/pulse IL-2 in the local tumor microenvironment. Additional studies have now been initiated to investigate the use of IL-12/pulse IL-2 in children with neuroblastoma, a study being done in collaboration with the New Approached for Neuroblastoma Therapy (NANT) Consortium. Future studies are planned using IL-18-based approaches in children with neuroblastoma and adults with solid tumors such as renal cell carcinoma.